Association of MicroRNA-155rs767649 Polymorphism with Susceptibility to Preeclampsia

Preeclampsia (PE) is a multifactorial disorder. Several studies showed that micro RNAs may play a critical role in PE pathogenesis. We aimed to investigate for the first time the association of mir-155rs767649 polymorphism with PE. Eighty patients with preeclampsia and 80 normal subjects were enrolled in the study. Serum expression levels of mature mir-155were evaluated using real-time PCR, and mir-155 rs767649 (T/A) polymorphism was genotyped using TaqMan SNP genotyping. There was a significant difference between the expression level of mir-155 in cases (5.86 ± 3.11) in comparison with controls (0.58 ± 0.30) (P<0.0001). Also,the minor allele of rs767649 was significantly associated with increased risk of PE [Recessive model: adjusted Odds ratio (OR) = 5.240, 95% confidence interval (CI) = (1.999-13.733),P= 0.001]. There was a significant difference between different genotypes according to expression levels of mir-155 in PE (P<0.0001) with high expression levels in TA genotype (7.10 ± 3.11 ). Mir-155 may play a critical role in PE pathogenesis. The obtained data suggest that a minor allele of rs767649 might be a predisposing factor for PE.

. It represents about 2-5% of pregnancies worldwide, and causes 10% to 15% of maternal deaths (2).The placenta plays an important role in the initiation and progression of the disease  (5).Reported risk factors include maternal age, nulliparity, diabetes, and hypertension (6). The condition could be complicated by elevated liver enzymes, hemolysis and low platelet count syndrome or eclampsia with visual disturbances and seizures (7).
MicroRNAs (miRNAs) represent a subgroup of non-protein-coding RNAs that are short (about 22 nucleotides) and highly conserved. They are key regulators of gene expression by destabilizing mRNAs or down-regulating their target genes (8).
Evidence proved that mir-155 was up-regulated in the placenta from numerous pregnant women suffering from PE. Mir-155 can be significantly upregulated by tumor necrosis factor and lipopolysaccharide, and it can regulate nuclear factor (NF)-kB (9).
Single nucleotide polymorphisms (SNPs) are DNA sequence variations that can interfere with posttranscriptional activities such as protein binding, polyadenylation, and miRNA binding. So, they can affect gene regulation (10). The rs767649 polymorphism in the promoter of mir-155 was reported in many diseases such as cervical cancer (11), hepatocellular carcinoma, (12) and lung cancer (13).
We aimed to detect the expression level of mir-155 and the association of mir-155 rs767649 polymorphism with PE.

Samples collection
Six ml blood was withdrawn and collected in 3 tubes.One of them being a plain tube that was allowed to clot for 15 min, and centrifuged at 4000g for 10 min. Serum samples were separated and The sample size was calculated according to Epi Info2000, a special formula used based on the prevalence of disease at a confidence interval (CI) of 95% and a precision of 2%. The sample size was increased by 10% to overcome problems related to missing data.

RNA extraction and reverse transcription reaction
RNAs were extracted from all samples using (Qiagen, Germany) RNA extraction kit, and reverse transcribed into cDNAs using (Qiagen, Germany) RT-PCR kit according to manufacturer's instructions.

Real-time PCR
The serum expression level of mir -155 was The relative expression of RNA was calculated by the 2 -ΔΔCt method for relative quantification (14).

Genotyping
DNA was extracted from whole EDTA blood samples using the QIAamp DNA MiniKit (Qiagen, Germany). Mir-155 rs767649 (T/A) polymorphism was genotyped using TaqMan SNP Genotyping assay. DNA amplification was performed using a Rotor gene Q System (Qiagen, Germany).

Statistical analysis
Statistical analyses were performed with SPSS V 20. Demographic differences between groups were examined by Mann-Whitney U and Chisquared (χ2) test. The correlation of study parameters was examined by Spearman correlation.
The frequencies of the alleles and genotypes were analyzed by the (χ2) test. The odds ratio (OR) and 95 % confidence intervals (CI) were also estimated by using logistic regression analyses to evaluate the associations between genotypes and PE with adjustment for age and body mass index (BMI).
Data were presented as the median. A comparison between genotypes was done by Kruskal-Wallis and Chi-squared (χ2) test. The value of P < 0.05 was considered as statistically significant. Table 1 shows that there was a significant difference between subjects and controls regarding  Table 1 shows also that 40% of cases had a mild degree of the disease while 60% had a severe degree.

MiR-155 expression levels in preeclampsia patients
There was a significant difference between the cases and controls regarding the expression level of mir-155 with up-regulation in PE patients (5.86 ± 3.11) in comparison with controls (0.58 ± 0.30) (P<0.0001) ( Table 1).

Correlation of the expression levels of mir-155 with study parameters among cases
Spearman correlation among study parameters in cases showed that there was a positive correlation between the expression level of mir-155and age (r =0.002), gravity (r=0.006), parity (r=0.045), abortion (r=0.003), fetal birth weight (r=0.036) while it showed negative correlation with FBS (r=-0.036) ( Table 2).

Genotypes and alleles frequencies of rs767649
Logistic regression analysis revealed that the  Table 3).

Pregnancy and delivery characteristics for different rs767649 genotypes in preeclampsia cases
Comparison of pregnancy and delivery characteristics for different genotypes in cases showed that there were significant differences between different genotypes according to gestational age (P <0.0001), fetal birth weight (P = 0.013), intra uterine growth retardation (P= 0.003) ( Table 5). Table 6 showed that there were significant

Discussion
During normal pregnancy, the placenta expresses different miRNAs according to gestational age (15). A major source of placental miRNAs is the villous trophoblasts (16,17).
Hypoxia plays an important role in their activity (18). For example, mir-146a and mir-223 are known to be dysregulated in PE, and interact with many immune cells such as macrophages and dendritic cells (19).Mir-155 is another miRna that has been investigated widely in several immunological disorders (20). It is processed in found that mir-155 has been induced by toll-like receptors in macrophages, and acts as a target of many inflammatory mediators (25). Also, serum levels of mir-155 and interleukin-17A were found to increase in PE cases in comparison with controls (26).
In the current study, we found that there was a  (30).
Genetic variants in the functional elements of miRNAmay affect its expression, maturation or mRNA recognition, and alter disease susceptibility (31). Rs767649 T > A polymorphism of mir-155 has recently been studied in many diseases (11)(12)(13) and for the first time we searched its role in PE. The results showed that the minor allele of rs767649 was significantly associated with increased risk of PE,and A allele was significantly associated with PE risk, compared with T allele. Also, there were significant differences between different genotypes regarding mir-155 expression level in PE (P<0.0001) with a high level in TAgenotype.
Diagnostic performance analysis of mir-155 showed its diagnostic value to differentiate PE patients from healthy control subjects as follows: AUC = 0.950, P < 0.0001, cutoff point 1.57, 95.0% sensitivity, and 100.0% specificity (Figure 1) which revealed that the relative expression level of mir-155 could be used as a potential biomarker for PE diagnosis and prognosis, and also as a promising management tool.
In conclusion, mir-155 may play a critical role in PE pathogenesis. The obtained data suggest that